Cholesterol-producing yeast strains and uses thereof

ABSTRACT

The invention concerns the production of cholesterol in organisms of the Fungi kingdom. More particularly, the invention concerns genetically modified Fungus independently producing cholesterol from a simple carbon source. The invention also concerns the use of the inventive Fungus for producing non-marked and marked cholesterol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a §371 National Filing of International PatentApplication No. PCT/FR2005/001090, filed May 2, 2005, which claimspriority under 35 U.S.C. § 119(a) to French Patent Application No,04/04,890, filed on May 6, 2004, the disclosures of which areincorporated herein in their entirety.

The present invention relates to the production of cholesterol inorganisms of the kingdom Fungi.

Cholesterol (cf. FIG. 1) is the most important animal sterol. It is afundamental component of cell membranes, of which it controls thefluidity, and is present in all animal tissues and particularly innervous tissue.

Cholesterol is a product of considerable industrial interest. Thus, itis commonly used in the cosmetics industry. It is also used in thepharmaceutical industry, for example in drug delivery, and also in cellculture.

Cholesterol is also used in the industrial synthesis of vitamin D₃. Thisvitamin is subsequently used to supplement human food (in dairyproducts, for example) and animal food. Cholesterol is alsoadvantageously used as an additive in animal food, in particular in foodintended for farmed shrimp.

Currently, the vast majority of cholesterol that is marketed isextracted from animal tissue (a tiny amount is produced by chemicalsynthesis). Two major starting sources are used for the extraction ofcholesterol: spinal cord from cattle and lanolin, which is the naturalfat of sheep's wool.

The use of animal tissue as a starting product raises problems. Thus,the recent problems associated with transmission of the prionresponsible for sheep scrapie to cattle (disease called BSE (bovinespongiform encephalitis) in cattle) have recalled the need for care whenusing animal tissue as a starting material. However, despite the stepstaken, the risk of transmission of a pathogenic agent cannot be totallyexcluded. It would therefore be extremely advantageous to have a sourceof cholesterol that does not come from an animal tissue.

The aim of the present invention is to provide an abundant source ofcholesterol that is safe from a health point of view. The inventors haveshown, surprisingly, that it is possible to divert the naturalproduction of ergosterol in Fungi so as to produce cholesterol.

GENERAL DESCRIPTION OF THE INVENTION

A first aspect of the invention concerns an organism of the kingdomFungi that autonomously produces cholesterol.

A second aspect of the invention concerns an organism of the kingdomFungi as defined above, wherein the latter is genetically modified.

A third aspect of the invention concerns an organism of the kingdomFungi as defined above, wherein the latter produces cholesterol from asimple carbon source.

The invention also relates to an organism of the kingdom Fungi asdefined above, expressing the 7-dehydrocholesterol reductase and3β-hydroxysterol Δ24-reductase enzymes. More particularly, the inventionrelates to an organism as defined above, in which the sterol24-C-methyltransferase enzyme has been inactivated and/or the C-22sterol desaturase enzyme has been inactivated.

Another aspect of the invention concerns an organism of the kingdomFungi as defined above, wherein the expression of the7-dehydrocholesterol reductase and 3β-hydroxysterol Δ24-reductaseenzymes is obtained by transformation of the organism.

The invention also relates to an organism of the kingdom Fungi asdefined above, wherein the inactivation of the sterol24-C-methyltransferase enzyme is carried out by gene inactivation and/orthe inactivation of the C-22 sterol desaturase enzyme is carried out bygene inactivation.

Another aspect of the invention concerns an organism of the kingdomFungi as defined above, which is chosen from the phylum Ascomycetes,more particularly from the subphylum Saccharomycotina, even moreparticularly from the class Saccharomycetes or Schizosaccharomycetes,even more particularly from the order Saccharomycetales orSchizosaccharomycetales, even more particularly from the familySaccharomycetaceae or Schizosaccharomycetaceae, even more particularlyfrom the genus Saccharomyces or Schizosaccharomyces.

Another aspect of the invention concerns an organism of the kingdomFungi as defined above, which is a yeast of the species Saccharomycescerevisiae or Schizosaccharomyces pombe.

The invention also relates to a method for producing cholesterol ofnonanimal origin, comprising the culturing of an organism as definedabove. More particularly, in this method, the step consisting inculturing the organism is followed by a step consisting in extractingthe cholesterol. Preferably, the extraction of the cholesterol iscarried out with a non-water-miscible solvent.

More particularly, in the method as defined above, a saponification stepis carried out before the extraction of the cholesterol. Even moreparticularly, in the method as defined above, a step consisting inmechanical grinding of the cells is carried out before thesaponification or the extraction of the cholesterol.

Another aspect of the invention concerns the use of an organism of thekingdom Fungi as defined above, for producing cholesterol, or one of itsmetabolic intermediates, or a mixture of sterols, labeled with ¹³C orwith ¹⁴C

The invention also relates to a method for producing cholesterol, or oneof its metabolic intermediates, or a mixture of sterols, labeled with¹³C or with ¹⁴C, comprising the following steps:

-   -   culturing an organism of the kingdom Fungi as defined above on a        ¹³C-labeled or ¹⁴C-labeled substrate, and    -   extracting said cholesterol, or one of its metabolic        intermediates, or the mixture of sterols.

The invention also relates to a method for producing an isotopic mixtureof cholesterol, of cholesterol intermediates or of cholesterolmetabolites, labeled at various positions using isotope labels,comprising culturing an organism of the kingdom Fungi as defined aboveon a labeled substrate and then on an unlabeled substrate, the culturetimes on each of these substrates being chosen in order to obtain adefined isotope profile. The invention also relates to a sample ofmolecules of cholesterol, of cholesterol intermediates or of cholesterolmetabolites labeled at various positions using isotope labels, that hasa defined isotope profile and that can be obtained by means of thismethod of production.

The invention also relates to a composition containing, as atraceability label, an isotopic mixture of cholesterol, of cholesterolintermediates or of cholesterol metabolites, labeled at variouspositions using isotope labels and having a defined isotope profile.More particularly, this composition is intended for the field of humanor animal food or therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the production of cholesterol inorganisms of the kingdom Fungi. In Fungi, no cholesterol is found in thenatural state, the latter being an animal sterol. The major sterol ofthe cell membranes of these organisms is ergosterol.

The present invention makes it possible to perform cholesterolsynthesis, through the multiplication of Fungi, in the presence of asimple carbon source. The method proposed by the present inventiontherefore makes it possible to obtain a large amount of cholesterol, atlow cost, since the method uses the culturing of organisms of thekingdom Fungi and the addition of a simple carbon source, readilyavailable commercially.

According to the present invention, the term “simple carbon source” isintended to mean carbon sources that can be used by those skilled in theart for the normal growth of a fungus and in particular of a yeast. Itis intended to denote in particular the various assimilable sugars, suchas glucose, galactose or sucrose, or molasses, or the by-products ofthese sugars. A simple carbon source that is most particularly preferredis ethanol and glycerol.

The fact that the production is carried out autonomously means thatthere is no need to add substrates in order to obtain the cholesterol,but that the organism can produce it only from the starting simplecarbon source. It is also clear that the strain can produce thecholesterol using a substrate located upstream in the metabolic pathway,insofar as the strain of the organism according to the present inventioncontains all the genes required to complete the metabolic pathway forcholesterol production.

The invention relates in particular to a genetically modified organismof the kingdom Fungi (a Fungus) that autonomously produces cholesterolfrom a simple carbon source.

A certain number of genetic modifications of the fungus can be effectedin order to divert the natural metabolic pathway of ergosterolproduction toward the production of cholesterol. The present inventionthus relates to a genetically modified organism of the kingdom Fungiexpressing the 7-dehydrocholesterol reductase and 3β-hydroxysterolΔ24-reductase enzymes. The strain of organism of the kingdom Fungi thusmodified produces cholesterol. The Applicant has in fact been able tomodel, by virtue of the results obtained (cf. the example section of thepresent application), the metabolic pathway resulting in ergosterol andin some of its derivatives (cf. FIG. 2). Expression of the7-dehydrocholesterol reductase and 3β-hydroxysterol Δ24-reductaseenzymes in the fungus S. cerevisiae can allow the production ofcholesterol by diverting part of the biosynthetic pathway forergosterol.

The 7-dehydrocholesterol reductase enzyme bears the number EC: 1.3.1.21in the International Enzyme Classification. It is also calleddelta-5,7-sterol-delta-7-reductase, 7-DHC reductase or Steroldelta-7-reductase, and will also be called Delta-7 sterol reductase,Delta-7Red, Delta 7 Reductase or Δ7-reductase in the remainder of thisdocument. This enzyme catalyzes, in the natural state in plants, forexample the NADPH-dependent reduction of delta-5,7-cholestadienol todelta-5-cholestaenol or the reduction of sterol intermediates having thedouble bond in the 7-8 position (Taton and Rahier, 1991). The geneencoding the 7-dehydrocholesterol reductase enzyme was isolated for thefirst time in the plant Arabidopsis thaliana; the isolation of thecorresponding gene and the expression of this enzyme in the yeastSaccharomyces cerevisiae is described in patent EP 727 489. Thesequences of this gene and of the protein are accessible under thefollowing GenBank accession number: U49398 (Lecain et al., 1996).

A certain number of homologues of this gene have been described in otherspecies. These are, for example, the homologous gene in humans (thenucleotide sequence of which is accessible under GenBank numberAF034544, the protein sequence of which is accessible under GenBanknumber: AAC05086) (Moebius et al., 1998); the homologous gene in the ratRattus norvegicus (the nucleotide sequence of which is accessible underGenBank number: AB016800, the protein sequence of which is accessibleunder GenBank number: BAA34306). Homologous genes have also beenidentified in the chicken Gallus gallus, with the Genbank referenceBM490-402 or in the toad Xenopus laevis, with the Genbank referenceBI315007, or the zebra fish Danio rerio, with the Genbank referenceBQ132664. A gene encoding a delta7 sterol reductase activity is alsofound in plants such as rice, Oryza sativa, with the Genbank referenceCA753545, or potato, Solarium tuberosum, with the Genbank referenceBF342071. This gene encoding a delta7 sterol reductase activity can alsobe found in the protist Mastigamoeba balamuthi, with the Genbankreference BE636562.

Those skilled in the art will be able to readily isolate otherhomologous genes encoding the 7-dehydrocholesterol reductase enzyme inother organisms. They may in particular refer to the cloning methoddescribed in example 1 of patent EP 727 489, which describes a cloningmethod for isolating a cDNA encoding a protein havingdelta-5,7-sterol-delta-7-reductase activity. Those skilled in the artmay also readily determine the 7-dehydrocholesterol reductase activityof the corresponding proteins, in particular using the activity assayalso described in example 1 of patent EP 727 489.

Expression of the 7-dehydrocholesterol reductase enzyme in an organismof the kingdom Fungi according to the invention can be obtained by anymeans known to those skilled in the art. This may in particular involvetransformation of the organism with a construct comprising an expressioncassette consisting of a transcription promoter, preferably homologous,of the open reading frame encoding the 7-dehydrocholesterol reductaseenzyme and of a suitable transcription terminator, according to theusual rules known to those skilled in the art. As homologous promoter,use will in general be made of a promoter that is suitable for allowingsufficient and functional expression of the heterologous protein. Thepromoter may, for example, be the PGK promoter, the ADH promoter, theCYC1 promoter, the GAL10/CYC1 promoter, the TDH3 promoter or the TPIpromoter. The terminator may, for example, be the terminator of thephosphoglycerate kinase (PGK) gene. Said expression cassette can beintegrated, in the form of one or more copies, into the nuclear ormitochondrial genome of the host, or can be carried by an artificialstructure of the yeast artificial chromosome (YAC) type or be carried byan episomal genetic element such as a plasmid. In order to effect thistype of expression, yeast of the Yarrowia lipolitica, Kluyveromyceslactis or Pichia pastoris type can, for example, be used.

Preferably, the 7-dehydrocholesterol reductase enzyme expressed is theenzyme of the plant Arabidopsis thaliana (an example of method ofexpression of this enzyme in the yeast Saccharomyces cerevisiae isdescribed in patent EP 727 489). It may, however, be any homologous ornonhomologous, natural or artificial, enzyme exhibiting the same enzymeactivity.

The 3β-hydroxysterol Δ24-reductase enzyme, also called DHCR24 or24-dehydrocholesterol reductase, naturally catalyzes the reduction ofdesmosterol (cholesta-5,24-dienol) or of lanosterol derivatives having adouble bond in the 24-25 position on the side chain (for example,14-desmethyl-lanosterol, zymosterol or cholesta-7,24-dienol), whichreduction is necessary for the biosynthesis of cholesterol in humans inparticular (H R. Waterham et al., 2001). This enzyme will also be calleddelta 24-(25) sterol reductase, delta 24 sterol Reductase orΔ24-reductase in the remainder of this document.

The gene encoding the 3β-hydroxysterol Δ24-reductase enzyme was isolatedfor the first time in humans; the isolation of the corresponding geneand the expression of this enzyme in the yeast Saccharomyces cerevisiaeis described in the publication H R. Waterham et al., 2001. Thesequences of this gene and of the protein are accessible under thefollowing GenBank accession numbers: NM_(—)014762 and NP_(—)055577.

A certain number of homologues of this gene have been described in otherspecies. They are, for example, the homologous gene in mice (Musmusculus) (the nucleotide sequence of which is accessible under GenBanknumber: NM_(—)053272, the protein sequence of which is accessible underGenBank number: NP_(—)444502). Homologues have been described in theworm Caenorhabditis elegans, and in particular a complementary DNA withthe Genbank reference AF026214. Homologous sequences have also beendescribed in plants, such as cotton, Gossypium hirsutum, with theGenbank reference AAM 47602.1, rice, Oryza sativa, with the Genbankreference AAP53615, or pea, Pisum sativum, with Genbank referenceAAK15493.

Those skilled in the art will be able to readily isolate otherhomologous genes encoding the 3β-hydroxysterol Δ24-reductase enzyme inother organisms. They may in particular refer to the cloning methoddescribed in the publication H R. Waterham et al., 2001. Those skilledin the art will also be able to readily determine the 3β-hydroxysterolΔ24-reductase activity of the corresponding proteins, in particularusing the activity assay also described in the publication (Waterham etal., 2001).

Expression of the 3β-hydroxysterol Δ24-reductase enzyme in an organismof the kingdom Fungi according to the invention can be obtained by anymeans known to those skilled in the art. This may in particular involvethe means described above with regard to the expression of the7-dehydrocholesterol reductase enzyme.

Preferably, the 3β-hydroxysterol Δ24-reductase enzyme expressed is thehuman enzyme. An example of isolation of the corresponding gene and ofexpression of this enzyme in the yeast Saccharomyces cerevisiae isdescribed in the publication H R. Waterham et al., 2001. It may,however, be any homologous or nonhomologous, natural or artificial,enzyme exhibiting the same enzyme activity.

Advantageously, the organisms of the kingdom Fungi according to thepresent invention express the 7-dehydrocholesterol reductase and3β-hydroxysterol Δ24-reductase enzymes and also exhibit inactivation ofthe sterol 24-C-methyltransferase enzyme.

The sterol 24-C-methyltransferase enzyme bears the number EC-2.1.1.41 inthe International Enzyme Classification. It is also called ERG6p,Delta(24)-methyltransferase, Delta(24)-sterol methyltransferase,Zymosterol-24-methyltransferase, S-adenosyl-4-methionine:steroldelta(24)-methyltransferase, SMT1, 24-sterol C-methyltransferase,S-adenosyl-L-methionine:delta(24(23))-sterol methyltransferase orPhytosterol methyltransferase. This enzyme naturally catalyzes the C-24methylation of zymosterol, resulting in the formation of fecosterol.

The gene encoding the sterol 24-C-methyltransferase enzyme was namedErg6 in the yeast Saccharomyces cerevisiae. The sequence of this gene isaccessible under the following GenBank accession number: NC_(—)001145.The sequence of the corresponding protein is accessible under thefollowing GenBank accession number: NP_(—)013706 (Bowman et al., 1997),(Goffeau et al., 1996).

A certain number of homologues of this gene have been described in otherFungi. They are, for example, the homologous gene in Schizosaccharomycespombe (the nucleotide sequence of which is accessible under GenBanknumber Z99759, the protein sequence of which is accessible under GenBanknumber: CAB16897) (Wood et al., 2002); the homologous gene in Neurosporacrassa (the nucleotide sequence of which is accessible under GenBanknumber: NCB24P7, the protein sequence of which is accessible underGenBank number: CAB97289); the homologous gene in Candida albicans (thenucleotide sequence of which is accessible under GenBank number:AF031941, the protein sequence of which is accessible under GenBanknumber: AAC26626) (Jensen-Pergakes et al., 1998). Genes encoding anenzyme homologous to ERG6 have also been described in Candidalusitaniae, with Genbank reference AA021936.1 and also in Pneumocystiscarinii (Kaneshiro et al., 2002) or in Kluveromyces lactis(Ozier-Kalogeropoulos et al., 1998).

Those skilled in the art will be able to readily isolate other geneshomologous to the Erg6 gene in organisms of the kingdom Fungi. Thoseskilled in the art will also be able to readily determine the sterol24-C-methyltransferase activity of the corresponding proteins, inparticular using, as activity assay, the functional complementation of ayeast strain disrupted for these genes. The complementation is thenattested to by the formation of sterols that are branched at the24-position, in particular of sterols of ergosta-type carrying amethylene group at the 24-28 position. The presence of ERG6-type sterol24-C-methyltransferase biological activity will also be determined invitro by means of the techniques developed by (McCammon et al., 1984) orby Taylor and Parks (Taylor and Parks, 1978). Furthermore, the sterolsproduced and the substrate for the ERG6 enzyme will be separated by gaschromatography according to the technique developed by Nes in (Methodsin Enzymology Steroids and Isoprenoids Volume 111 part B, 1985, “Acomparison of Methods for the Identification of Sterols”, pp. 3-37).

The strain of organism of the kingdom Fungi according to the presentinvention expressing the 7-dehydrocholesterol reductase and3β-hydroxysterol Δ24-reductase enzymes and also exhibiting inactivationof the sterol 24-C-methyltransferase enzyme produces cholesterol. TheApplicant has in fact been able to determine that, surprisingly, theinactivation of the sterol 24-C-methyltransferase enzyme blocks thebiosynthetic pathway for ergosterol upstream, and allows increasedproduction of cholesterol by the fungus strain (cf. the example sectionof the present application).

The 7-dehydrocholesterol reductase and 3β-hydroxysterol Δ24-reductaseenzymes are expressed as described above.

The inactivation of the sterol 24-C-methyltransferase enzyme can becarried out by any means known to those skilled in the art. It may inparticular involve the introduction, by mutagenesis, of a nonsensemutation, of an insertion or of a deletion that causes a change in thereading frame in the gene encoding said protein.

It may also involve the expression of an antisense RNA that iscomplementary to the messenger RNA encoding said protein, or the genesilencing system known to those skilled in the art as RNAi (smallinterfering RNA) and the associated enzyme systems if these do notnaturally exist in the host. The mutagenesis can be effected in thecoding sequence or in a noncoding sequence so as to render the encodedprotein inactive or to prevent its expression or its translation. Themutagenesis can be effected in vitro or in situ, by suppression,substitution, deletion and/or addition of one or more bases in the geneunder consideration, or by gene inactivation.

This may in particular involve the introduction of an exogenous DNA intothe coding sequence or promoter sequence (for example an expressioncassette with homologous promoter and/or terminator and a heterologouscoding portion). The expression cassette advantageously allows theexpression of a selection marker. It is also possible to modify thepromoter of the gene in order to reduce the level of expression. Forfungi, inactivation is also carried out by interruption of the codingsequence with the coding sequence of a heterologous or homologous markergene. The main techniques for interrupting a gene from fungi aredescribed in the article by Johnston et al., (2002) (Methods inEnzymology Volume 350 Edited by Christine Guthrie and Gerry Fink; “GeneDisruption”; M. Johnston, L. Riles, J. Hegemann, pp. 290-315).

Advantageously, the organisms of the kingdom Fungi according to thepresent invention express the 7-dehydrocholesterol reductase and3β-hydroxysterol Δ24-reductase enzymes and also exhibit inactivation ofthe C-22 sterol desaturase enzyme.

The C-22 sterol desaturase enzyme is also called ERG5p, Cyp61,cytochrome p-45061 or sterol delta22-desaturase. This enzyme naturallycatalyzes the conversion of ergosta-5,7,24(28)-trienol toergosta-5,7,22,24(28)-tetraenol by adding a double bond at position C22(cf. FIG. 2).

The gene encoding the C-22 sterol desaturase enzyme was named Erg5 inthe yeast Saccharomyces cerevisiae. The sequence of this gene isaccessible under the following GenBank accession number: U34636. Thesequence of the corresponding protein is accessible under the followingGenBank accession numbers: AAB06217 (Skaggs et al., 1996) or P54781(Bowman et al., 1997).

A certain number of homologues of this gene have been described in otherFungi. They are, for example, the homologous gene in Schizosaccharomycespombe (the nucleotide sequence of which is accessible under GenBanknumber Z98974, the protein sequence of which is accessible under GenBanknumber: CAB11640) (Wood et al., 2002); the homologous gene inSymbiotaphrina buchneri (the nucleotide sequence of which is accessibleunder GenBank number: AB086896, the protein sequence of which isaccessible under GenBank number: BAC01142) (Noda and Koizumi, 2003); thehomologous gene in Symbiotaphrina kochii (the nucleotide sequence ofwhich is accessible under GenBank number: AB086890, the protein sequenceof which is accessible under GenBank number: BAC01139) (Noda andKoizumi, 2003); the homologous gene in Candida albicans (the nucleotidesequence of which is accessible under GenBank number: AL033396, theprotein sequence of which is accessible under GenBank number: CAA21953)(Tait et al., 1997). The ERG5 gene has also been described in Candidalusitaniae, with Genbank reference AA048601.

Those skilled in the art will be able to readily isolate other geneshomologous to the Erg5 gene in organisms of the kingdom Fungi. Thoseskilled in the art will also be able to readily determine the C-22sterol desaturase activity of the corresponding proteins, in particularusing the activity assay described by B. A. Skaggs et al., 1996. Thisactivity may also be demonstrated by functional complementation of an S.cerevisiae yeast disrupted beforehand in the erg5 gene. Thiscomplementation will be attested to by the presence, in the complementedstrain, of ergosta-5,7,22-trienol. The C22 sterol desaturase activitycan be measured in vitro using the method described by Kelly and Baldwinet al., JBC (1997), after lysis of the yeast (Kelly et al., 1997).

The strain of organism of the kingdom Fungi according to the presentinvention expressing the 7-dehydrocholesterol reductase and 3(3-hydroxysterol Δ24-reductase enzymes and also exhibiting inactivationof the C-22 sterol desaturase enzyme produces cholesterol. The Applicanthas in fact been able to determine that the inactivation of the C-22sterol desaturase enzyme advantageously blocks the conversion ofcholesterol to cholesta-5,22-dienol and allows stabilization of theproduction of cholesterol (cf. the example section of the presentapplication). This blockage also occurs at the level of the conversionof cholesta-5,7-dienol, a precursor of cholesterol, tocholesta-5,7,22-trienol, a precursor of cholesta-5,22-dienol.Surprisingly, the C-22 sterol desaturase enzyme in fact acceptscholesterol as a substrate, and converts it to cholesta-5,22-dienol.This parasitic reaction can be eliminated by inactivating the C-22sterol desaturase enzyme, as the Applicant has been able to determine.

The expression of the 7-dehydrocholesterol reductase and3β-hydroxysterol Δ24-reductase enzymes is carried out as describedabove. The inactivation of the C-22 sterol desaturase enzyme can becarried out by any means known to those skilled in the art. They may inparticular be the methods described above with regard to theinactivation of the sterol 24-C-methyl-transferase enzyme.

Advantageously, the organisms of the kingdom Fungi according to thepresent invention express the 7-dehydrocholesterol reductase and3β-hydroxysterol Δ24-reductase enzymes and also exhibit inactivation ofthe C-22 sterol desaturase enzyme and inactivation of the sterol24-C-methyltransferase enzyme. These strains in fact exhibit thecumulative advantages of the two inactivations and arecholesterol-producing strains.

The expression of the 7-dehydrocholesterol reductase and3β-hydroxysterol Δ24-reductase enzymes and the inactivation of the C-22sterol desaturase and sterol 24-C-methyltransferase enzymes are carriedout as described above.

In one embodiment, the cholesterol is present in the strain of organismaccording to the present invention in a proportion greater than 20%,preferably 35%, most preferably 50% or more of the total sterolsproduced by the strain according to the invention (in particular thesynthesis intermediates).

Preferably, the organisms of the kingdom Fungi according to the presentinvention are chosen from the phylum Ascomycetes, more preferably theyare chosen from the subphylum Saccharomycotina, even more preferablythey are chosen from the class Saccharomycetes or Schizosaccharomycetes,even more preferably they are chosen from the order Saccharomycetales orSchizosaccharomycetales, even more preferably they are chosen from thefamily Saccharomycetaceae or Schizosaccharomycetaceae, even morepreferably they are chosen from the genus Saccharomyces orSchizosaccharomyces, entirely preferably, the organisms of the kingdomFungi according to the invention belong to the species Saccharomycescerevisiae or Schizosaccharomyces pombe.

The present invention also relates to a method for producing cholesterolof nonanimal origin, comprising the following steps:

-   -   an organism of the kingdom Fungi as defined above is cultured,    -   the cholesterol produced by this organism is extracted.

The extraction is based on the treatment of the fungus with a solventfor cholesterol, preferably a non-water-miscible solvent. This treatmentcan preferably be combined with any method of mechanical grinding of thecells. More preferably, the fungus will be treated, before extractionwith the solvent, with a saponification mixture intended to release thecholesterol possibly bound to other cellular components such as, inparticular, fatty acids. This saponification mixture may consist of abase, for example aqueous ammonia, sodium hydroxide or potassiumhydroxide, dissolved in water or, more preferably, in a water-miscibleorganic solvent such as, for example, methanol or ethanol, or asolvent-water mixture. The saponification may be carried out without orpreferably with heating to a temperature of 60-120° C., at atmosphericpressure or at low pressure. The extraction with the non-water-misciblesolvent may be replaced with a solid-phase extraction on a hydrophobicresin. A sterol extraction method is described by L. Parks et al.,(1985) (Methods in Enzymology 111 Edited by L. Rilling, L. Parks, C.Bottema, R. Rodriguez and Thomas Lewis, pp. 333-339).

The crude cholesterol thus obtained may be purified by any methods knownto those skilled in the art, in particular that described by Boselli E,Velazco V, Caboni Mf and Lercker G J, Chromatogr A. 2001 May 11; 917(1-2):239-44.

Other methods may also be used, such as that described for theextraction of cholesterol from sheep's wool. Those skilled in the artmay in particular refer to the methods described in American patentsU.S. Pat. No. 2,688,623 or U.S. Pat. No. 2,650,929, or in Britishpatents GB690879, GB646227 or GB613778.

Another aspect of the invention concerns the use of the strainsaccording to the present invention in order to obtain cholesterol or oneof its metabolic intermediates, or a labeled mixture of sterols. Theterm “metabolic intermediate of cholesterol” is intended to mean inparticular the sterols specified in FIG. 2. They may in particular becholesta-8,24(25)-dienol, cholesta-7,24(25)-dienol,cholesta-5,7,24(25)-trienol, cholesta-5,24(25)-dienol orcholesta-5,22-dienol.

The principle for obtaining a labeled cholesterol is described in FIG.10. This manipulation consists in first of all growing the fungus strainon a completely labeled substrate. The cells are then cultured on anunlabeled substrate. There is thus a change in isotope labeling of thecarbon source; there ensues de novo synthesis of metabolic intermediatesand then of sterol, including cholesterol, and comprising a gradualchange in labeling. This therefore involves a profile that is complexbut can be entirely experimentally determined, and that represents aunique isotope signature that depends at the same time:

-   -   1) on the labeling protocol and in particular on the culture        times and conditions with labeled and unlabeled substrate,    -   2) on the precise genetic structure of the strain used,    -   3) on the precise time at which the cultures are stopped.

Once the culture has been stopped (for example by cell lysis or bystopping the culture in the presence of a sublethal concentration ofcytotoxic or cytostatic antifungal products), the labeled cholesterol orone of its metabolic intermediates, or a labeled mixture of sterols, isextracted and purified as described above.

The isotope profile of the labeled cholesterol or of one of itsmetabolic intermediates, or of the labeled mixture of sterols, hasseveral unique properties:

-   -   1) it can be modulated as desired by adjusting the culture        conditions, the strain used and the sterol chosen. A unique        label register can therefore be produced;    -   2) it is “combinable”, i.e. several isotope signatures        corresponding to several unique sterols labeled with isotope        profiles that can themselves be modulated can be combined so as        to form a “molecular alphabet”;    -   3) it is reproducible and easy to determine experimentally;    -   4) it corresponds to a molecular tracer mixture that is easy to        isolate, stable, colorless and odorless, nonvolatile and        nontoxic, and that can be incorporated into foods, a medicinal        product, additives or other products that can be assimilated by        humans;    -   5) it cannot be falsified without having the specific        recombinant strains and the very precise labeling, culturing and        extraction conditions. In addition, knowledge of the isotope        signature does not make it possible to track back to the        parameters which made it possible to produce it.

Thus, an “isotope alphabet” for general use, that cannot be falsifiedand that can be incorporated into products of any type, includingconsumables, can be readily obtained by virtue of the present invention.There is a virtually unlimited number of “isotope words” that can beconstituted from such an alphabet by making use of both the labelingprofiles and the various types of sterols. The incorporation of suchsignatures into the most varied products therefore constitutes a uniquemethod of labeling that cannot be falsified, unlike, for example, DNAsignatures, which can be reproduced once they are known. The signaturecan, moreover, be read nondestructively, for example by laser ionizationfollowed by mass spectrometry analysis (MALDI-TOF or the like).

The use of ¹³C-labeled substrate instead of the unlabeled carbon sourcesfor culturing the fungus strains according to the invention makes itpossible to synthesize very highly labeled sterols, and in particularcholesterol (comprising at least 95% of ¹³C carbon). The preparation of¹⁴C radioactive sterols and cholesterol is also possible by the sameapproach. The method can also be incorporated into yeast strains thatproduce steroids, and in particular hydrocortisone (cf. patentapplication WO 02/061109), so as to produce ¹³C-labeled or ¹⁴C-labeledsteroids, for example for RIA assays.

LEGEND FOR THE FIGURES

FIG. 1: Chemical formula of cholesterol, and also the nomenclaturegenerally used for numbering the various carbons and the name of thevarious rings. The four rings of the cholesterol molecule are named A,B, C and D, respectively, and the carbons are numbered from 1 to 27.

FIG. 2: Simplified scheme of the late portion of the biosyntheticpathway for sterols of the ergosta- and cholesta-types in natural ormodified yeast. The scheme is not exhaustive, but makes it possible todefine the steps involving the enzymes mentioned in this document. TheERG2p, ERG3p, ERG5p and ERG6p proteins are fungus or yeast proteins,whereas the Delta-7Red (Delta-7 sterol reductase) and Delta 24-(25)Red(Delta 24-(25) sterol reductase) proteins are heterologous proteins ofmammalian origin or of plant origin.

FIG. 3: Compared HPLC profile, with UV detection at 206 nm, of the freesterols of the strains derived from the BMA64 strain and identificationof these sterols. The strains studied are as follows: WGIF01 (BMA64strain disrupted in the erg6 gene (cf. example 1)), WGIF02 (BMA64 straindisrupted in the erg6 gene and expressing the Δ24-reductase, example12), WGIF03 (BMA64 strain disrupted in the erg6 gene and expressing theΔ7-reductase, example 13), WGIF04 (BMA64 strain disrupted in the erg6gene and expressing the Δ7-reductase and the Δ24-reductase, example 14).C5: cholesta-5-enol (cholesterol); C5,22: cholesta-5,22-dienol; C5,24:cholesta-5,24-dienol (desmosterol); C8,24: cholesta-8,24-dienol(zymosterol); C5,7,22: cholesta-5,7,22-trienol; C5,7,24:cholesta-5,7,24-trienol; C5,22,24: cholesta-5,22,24-trienol; C5,7,22,24:cholesta-5,7,22,24-tetraenol; lan: lanosterol.

FIG. 4: Compared HPLC profile, with UV detection at 206 nm, of the freesterols of the WGIF04 strain (BMA64 strain disrupted in the erg6 geneand expressing the Δ7-reductase and the Δ24-reductase, example 14) after0, 2, 4, 8 and 24 hours of induction with galactose. Δ: WGIF01 strain(example 1). For the WGIF04 strain, the samples are taken 0, 2, 4, 8 and24 h after switching of the carbon source to galactose. The profile forthe BMA64 strain bearing the erg6 disruption (WGIF01) presented is thatobtained immediately after the switch to galactose. This profile remainsvirtually unchanged during the induction (0-24 h). The absorption signalat 206 nm corresponds to absorption coefficients that are variable fromone sterol to the other. C5: cholesta-5-enol (cholesterol); C5,22:cholesta-5,22-dienol; C5,24: cholesta-5,24-dienol (desmosterol); C8,24:cholesta-8,24-dienol (zymosterol); C5,7,22: cholesta-5,7,22-trienol;C5,7,24: cholesta-5,7,24-trienol; C5,22,24: cholesta-5,22,24-trienol;C5,7,22,24: cholesta-5,7,22,24-tetraenol; lan: lanosterol.

FIG. 5: Compared HPLC profile, with positive ionization electrospraydetection (mass spectrometry), of the free sterols of the WGIF04 strain(example 14) after 0, 2, 4, 8 and 24 hours of induction with galactose.A: WGIF01 strain. C5: cholesta-5-enol (cholesterol); C5,22:cholesta-5,22-dienol; C5,24: cholesta-5,24-dienol (desmosterol); C8,24:cholesta-8,24-dienol (zymosterol). The HPLC profiles come from the sameassays as those of FIG. 4.

FIG. 5A (left): Detection at m/z= 367, FIG. 5B (right): m/z= 369.

y-axis: number of ions counted/second. x-axis: elution time in minutes.

FIG. 6: Details of the profile at m/z= 369 by HPLC for the threestrains: WGIF01, CA10 bearing the expression plasmid for delta 24 sterolreductase, and for WGIF04, cholesterol is injected as an internalstandard. The amounts of total sterols injected for the three strainscorrespond to extractions carried out on identical amounts of culturemeasured by the absorbance at 600 nm.

FIG. 7: Compared profiles of the total sterols (free and esters), by gaschromatography, of the WGIF01 (deletion of erg6), WGIF02 (deletion oferg6 with expression of the Δ24-reductase), WGIF03 (deletion of erg6with expression of the Δ7-reductase), WGIF04 (deletion of erg6 withexpression of the Δ24-reductase and A 7-reductase) and CA10 pYES_Delta24(FY1679 genetic background, deletion of erg5 with expression of theΔ24-reductase, Δ7-reductase, erg5) strains. The response scales (flameionization currents) are arbitrary. The profiles should only be comparedqualitatively from one strain to the other. The retention time scale is,however, the same for all the strains (the retention times are expressedin minutes). The sterols are identified according to the criteriadescribed in the present application.

FIG. 8: Quantitative distribution of the main free sterols in the yeaststrains (BMA64 (FIG. 8A), WGIF01 (FIG. 8B), WGIF02 (FIG. 8C) and WGIF03(FIG. 8D)) evaluated on the basis of the UV spectra. The distribution isgiven in % of the total species presented in the figure and which arethe only ones that can be detected in appreciable amounts. In theabsence of a standard for several of the intermediate sterols, thequantification is carried out on the basis of the UV spectra associatedwith each of the peaks of the HPLC chromatogram using the evaluatedabsorption coefficients given below (cf. table 1, the absorptioncoefficients are expressed in mM per liter and per cm.). To do this, theabsorption coefficients corresponding to the unsaturated structuralunits present in the structure of a given sterol are sought in table 1and optionally added (if several units are present in the same molecule)so as to provide an evaluation of the extinction coefficient of eachtype of sterol. The evaluation is done using the values at 280 nm if atleast one unit that is absorbent at this wavelength is present, failingthis, the wavelength 235 nm is used, and failing absorption at thelatter wavelength, the wavelength 206 nm is used to evaluate theconcentrations of each of the sterols from the respective absorptionsignals by HPLC.

FIG. 9: Quantitative distribution of the main free sterols in the WGIF4yeast strain, evaluated on the basis of the UV spectra. Thequantifications are carried out in the same manner as described in FIG.8.

FIG. 10: Principle of the isotope labeling of the sterols bysubstitution of carbon sources.

FIG. 11: Evaluation of the isotope labeling profiles for the cholesterolproduced in the WGIF04 strain after 4, 8 and 24 hours of induction. Thefree sterols are extracted and separated by HPLC as described. A massspectrum between the values of m/z 300 and m/z=450 is acquired every 0.2seconds during the elution. These spectra are then averaged over windowsof 1.8 seconds, and then subjected to a multilinear regression using, asregression base, a set of 24 vectors representing the theoretical massdistributions of the labeled cholesterol for a random incorporation, byindependent selection, of the carbon 13 at each of the 27 positions ofthe molecule with a probability of labeling on each carbon that isvariable between 0 and 1 according to the vector under consideration.The labeling probabilities for the various vectors used as base arechosen such that the cross-correlation coefficient for the distributionsof two consecutive vectors of the base is 0.92, the base beginning witha vector corresponding to a probability of presence of 100% at all thepositions on the carbon 12. The multilinear adjustment is made on thebasis of a least square statistical criterion, nullifying thenon-diagonal terms of the matrix of the products of 3 the partialderivatives of the Gauss method (maximum numerical filtering). Afteranalysis, the mass spectra are then reconstructed on the optimizedbasis. The curves represented in the figures therefore represent theresult of the optimal filtered reconstruction after standardization ofthe maximum amplitude at the value 100.

For each induction time, the two curves represent two independentprofiles corresponding to elution times that differ by 1.8 seconds andcorresponding to spectra located in the central zone of the cholesterolelution peak. The figure demonstrates that the analysis is highlyreproducible.

FIG. 12: Example of various isotope signatures with various sterols orvarious induction times. The same calculation and representation as forFIG. 11, but for various sterols and various induction times. The valueof RT indicates the range of retention time used for the calculation (inminutes). The values for this range are as follows:

FIG. 12 A: RT=12.25-12.42, FIG. 12 B: RT=12.2-12.7, FIG. 12 C:RT=12.25-12.35, FIG. 12 D: RT=13.3-13.6.

The induction times are 8 or 24 hours.The values of m/z indicate the left and right limits of the m/z values.The lowest value for m/z for each box corresponds to the m/z for thesterol made up entirely of carbon 12.

FIG. 13: Compared profiles of the total sterols (free and esters), ingas chromatography, of the YIM59/pIM303 strain (part A of the figure)and of the YIM59/pIM331 strain (part B of the figure) (cf. example 18).The response scales are arbitrary. The retention time scale is the samefor both strains (the retention times are expressed in minutes). Thesterols are identified according to the criteria described in thepresent application.

The present invention is illustrated using the following examples, whichshould be considered as nonlimiting illustrations.

The molecular biology techniques used are described by Ausubel et al.,some yeast manipulations are described by Adams et al. (Adams and Holm,1996).

EXAMPLE 1 Construction of an S. cerevisiae Yeast Strain with anInterruption in the Erg6 Gene (WGIF01 Strain)

The S. cerevisiae yeast strain WGIF01 in which the ERG6 gene isinterrupted with the TRP1 gene was obtained by transforming the BM64strain with a PCR product carrying a functional TRP1 gene bordered byextremities homologous to the ERG6 gene.

The BM64 strain (of genotype MATa; ura3-52; trp1Δ2; leu2-3_(—)112;his3-11; ade2-1; can1-100) is a derivative of the S. cerevisiae yeaststrain W303 MATα by complete deletion of the TRP1 gene. The BMA64 strainand the W303 MATα strain are described in the publication byBaudin-Baillieu et al. (Baudin-Baillieu et al., 1997).

To isolate the TRP1 gene, the TRP1 gene of the plasmid pFL44 (Bonneaudet al., 1991) was amplified using Z-TaqI (a DNA-dependent DNApolymerase) provided by the company Takara (PanVera LLC 501 CharmanyDrive, Madison, Wis. 53719 USA). The pair of primers used makes itpossible to amplify, by means of the DNA polymerase, the TRP1 genebordered by sequences corresponding to the ERG6 gene.

The sequence of these primers is as follows:

(SEQ ID No. 1) OERG6trp1: 5′(CCTAGCGACGAAAAGCATCATTGGAGTGAATAACTTGGACTTACCAttc ttagcattttgacg) 3′.(SEQ ID No. 2) OERG6trp2: 5′ 5′(GCATAAGAGTGAAACAGAATTGAGAAAAAGACAGGCCCAATTCAaattc gggtcgaaaaaagaaaagg)3′.

The PCR (polymerase chain reaction) product thus obtained is purified byelectroelution of the fragment corresponding to the expected size, andis used to transform the BM64 strain by the lithium chloride techniqueas described by (Gietz et al., 1995).

After transformation, the treated yeasts are plated out on a minimummedium containing no tryptophan (Gietz et al., 1995). 41 transformedBM64 colonies that are prototrophic for tryptophan are thus obtained.These 41 colonies are then tested for three of their properties:sensitivity to nystatin, genomic structure of the insertion of the TRP1gene, and profile by gas chromatography of the total sterols that theyproduce.

For this, the 41 colonies are transferred onto a minimum mediumcontaining, respectively, 10, 20 or 50 μg/ml of nystatin; about tencolonies are capable of growing on the medium containing a dose of 50g/ml of nystatin. These resistant colonies are selected in order toverify their gene structure and also their sterol compositions.

The insertion of the TRP1 gene into the ERG6 gene is verified by PCRusing a pair of oligonucleotides covering the junction between thefunctional TRP1 gene and the disrupted ERG6. This pair ofoligonucleotides is as follows:

(SEQ ID No. 3) OERG6trp3: AGGGCCGAACAAAGCCCCGATCTTC and (SEQ ID No. 4)OERG6trp4: GGCAAACCGAGGAACTCTTGG.

Some strains exhibit the expected PCR profile, i.e. a fragment of 800base pairs corresponding to the size expected for a TRP1 insertion intoERG6.

With the aim of verifying that the ERG6 gene is indeed inactivated inthese strains, an analysis of the sterol compositions of these strainsby gas chromatography and by high pressure liquid chromatography wascarried out (Duport et al., 2003; Szczebara et al., 2003).

These analyses confirm the absence of ergosterol synthesis and theaccumulation of abnormal sterols compatible with the expecteddisruptions of the biosynthetic pathway in the disrupted strain.

One strain was more particularly selected, and called WGIF01.

EXAMPLE 2 Construction of the CA10, CA14 and CA23 Strains

The CA10 strain (of genotype: MATα, rho⁺, GAL2, ura3-52, trp1-Δ63,his3-Δ200, erg5::HYGRO^(R), ade2::GAL10/CYC1::Δ7Reductase::PGK1,LEU2::GAL10/CYC1::matADR::PGK1), the CA14 strain (of genotype: MATα,rho⁺, GAL2, ura3-52, trp1-Δ63, his3-Δ200, erg5::HYGRO^(R)atf2::G418^(R), ade2::GAL10/CYC1::Δ7Reductase::PGK1,LEU2::GAL10/CYC1::matADR::PGK1), and the CA23 strain (of genotype: MATα,rho⁺, GAL2, ura3-52, trp1-Δ63, his 3-Δ200, erg5::HYGRO^(R),are1::G418^(R), are2::HIS3, ade2::GAL10/CYC1::Δ7Reductase::PGK1,LEU2::GAL10/CYC1::matADR::PGK1), and also the constructions thereof, aredescribed in the reference Duport et al., the technical content ofwhich, regarding the construction of these strains is incorporated intothe present application by way of reference.

These strains produce and contain, in their membranes, unnatural sterols(as described in European patent application EP 0727 489) and inparticular ergosta-5-enol (campesterol).

These three strains do not express the product of the ERG5 gene, whichis nonfunctional due to insertion into its coding sequence of thehygromycin resistance gene. In addition, these strains express the cDNAencoding plant Δ7 reductase (European patent application EP 0727 489describes in particular the cloning of the Δ7 reductase of the plantArabidopsis thaliana, which is incorporated into the present applicationby way of reference, the GenBank accession number of this sequence isATU49398).

The CA14 strain is derived from the CA10 strain by disruption of theATF2 gene. The product of this gene results in acetylation ofpregnenolone on position 3 (as is described in patent applicationWO99/40203).

The CA23 strain is a strain derived from the CA10 strain by deletion ofthe ARE1 and ARE2 genes; the two proteins Are1p and Are2p areresponsible for the esterification of ergosterol (Sturley, 2000) andpossibly of cholesterol since they are homologous to the enzymeresponsible for the esterification of cholesterol in mammals (ACAT).

EXAMPLE 3 Construction of the Plasmid for Expressing the Δ24-25Reductase of Human Origin (Plasmid pYES_Delta24)

The construction of this plasmid was described by Waterham et al., 2001.The construction consisted in placing the cDNA encoding Delta 24 sterolreductase under the control of the pGAL1 promoter and of the tCYC1terminator in the vector pYES2 (Invitrogen SARL, Cergy Pontoise,France). This plasmid is an E. coli/S cerevisiae shuttle plasmid andcontains a 2 micron origin of replication and a URA3 gene, allowing itto replicate in yeast and making it easy to select the yeast transformedwith this plasmid.

In addition, the GAL1 promoter is galactose-inducible.

EXAMPLE 4 Construction of the Plasmid pAG1 Expressing A. thalianaExpressing Δ7-Reductase

A plasmid was constructed specifically for the expression of A. thalianaDelta 7-reductase, on a single-copy vector. The plasmid pAM1 was usedfor this construction. This plasmid, the construction of which isdescribed in PCT application WO 02/061109 (cf. example 9.b of saidapplication, which is incorporated into the present application by wayof reference), is an E. coli/S. cerevisiae shuttle plasmid based on anautonomous replication sequence and a centromere (ARS CEN). Theselection marker is the ADE2 gene. This plasmid is compatible, and cantherefore replicate at the same time as a plasmid based on a 2 micronorigin of replication. This plasmid in particular has a unique NotI sitefor cloning expression cassettes, as described in the PCT applicationabove.

This site was used to clone an A. thaliana Delta7-reductase expressioncassette originating from the CA10 strain. In fact, this expressioncassette is very effective and enables the CA10 strain, which is alsodisrupted for ERG5, to produce campesterol (ergosta-5-enol) as the majorsterol (Duport et al., 1998). The genomic DNA fragment of the CA10strain containing the delta7-reductase gene is amplified using thefollowing primers:

(SEQ ID No. 5) OSA72 5′ (TATATAGCGGCCGCTTTCGCTGATTAATTACCCCAG) 3′ (SEQID No. 6) OSA 77 5′ (TATATAGCGGCCGCGAGAAGTGACGCAAGCATCA) 3′.

The amplification is carried out on the genomic DNA of the CA10 strainprepared by the rapid phenol/chloroform extraction technique asdescribed by Adams et al. (Adams and Holm, 1996).

Fifty nanograms of CA10 genomic DNA are used as matrix for amplificationusing the primers OSA72 and OSA77. The Taq DNA polymerase and theenzymatic conditions came from the company Stratagene. The amplificationconditions were as follows: initial denaturation for 5 min at 95° C.,then subsequently, thirty cycles were carried out consisting of adenaturation for 30 s at 95° C., a hybridization for 30 s at 50° C. andthen an elongation for 1 min at 72° C. The reaction is terminated by afinal extension for 10 min at 72° C.

The PCR fragment is then digested with the NotI enzyme and purified onagarose gel and then cloned conventionally into the unique NotI site ofthe plasmid pAM1. The plasmid thus obtained was named pAG1.

It is a single-copy vector for expressing A. thaliana Delta7-Reductasein yeast, the Delta7-Reductase gene being placed under the control ofthe GAL10/CYC1 promoter (Lecain et al., 1996).

EXAMPLE 5 Extraction of the Free and Esterified Sterols in the Yeast,for the Analyses 1) Conditions for Extracting the Free and EsterifiedSterols in the Yeast (Procedure 1)

a) Conditions for extracting the free sterols:

The cell pellet is washed twice with 500 μl of deionized and filteredwater, in a glass tube.

The cells are then resuspended in 500 μl of water containing 0.5 mmglass beads, corresponding to 150 μl of liquid in the tube.

An extraction is carried out twice with 2 ml of 1,2-dichloroethane withvigorous agitation on a vortex for 10 minutes. After the firstextraction, the mixture of cells, glass beads and solvent is centrifugedfor 5 minutes at 1500 g for the purpose of separating the two phases.

The two organic fractions derived from the two successive extractionsare combined and dried under a stream of nitrogen for a few hours.

The sterol extract is suspended in 100 μl of acetonitrile in order forit to be analyzed by high performance liquid chromatography (HPLC)(Szczebara et al., 2003) or in 100 μl of hexane for the gaschromatography (GC) analyses (Duport et al., 2003).

b) Conditions for Extracting the Total Sterols: Saponification andExtraction of the Esterified Sterols, Qualitative Analysis Procedure 1:

The cell pellet is resuspended in 500 μl of purified water. 2 ml ofpotassium hydroxide KOH at 10% in methanol are added to this suspension.The mixture is heated for one hour at 60° C. in closed tubes. After theincubation, and once the tubes have returned to ambient temperature, themixture is extracted three times with 2 ml of hexane. Between eachextraction, the two phases are separated by centrifugation for 5 min at1500 g. After each extraction, the organic phase is transferred into anew tube, and the three organic phases are then combined and then driedunder a stream of nitrogen.

The sterol residue is resuspended in 100 μl of 100% acetonitrile inorder for it to be analyzed by high performance liquid chromatography(HPLC) (Szczebara et al., 2003) or in 100 μl of hexane for the gaschromatography (GC) analyses (Duport et al., 2003).

2) Conditions for Extracting the Free and Esterified Sterols in theYeast, for Qualitative Analysis (Procedure 2)

The strains are cultured in rich medium (10 g of bactopeptone per literand 10 g of yeast extracts per liter) with 2% of glucose as carbonsource in order to obtain 500 mg of lyophilized cells. These dried cellsare taken up in 3 ml of methanol (100%) containing 1 g of KOH and atrace of pyrogallol, and the mixture is then incubated for 45 minutes at90° C. After returning to ambient temperature, the sterols are extractedwith 5 ml of hexane. The organic phase is separated into three sampleshaving the same volume, and is dried under a stream of air. Two of thesamples of the extracted sterols are taken up in 100 μl of hexane forthe analyses by gas chromatography (GC) and gas chromatography/massspectrometry GC/MS, and the third sample is taken up in 150 μl ofmethanol for the high performance liquid chromatography (HPLC) studies.

EXAMPLE 6 Analysis of the Free and Esterified Sterols in the Yeast byGas Chromatography (GC)

1) Gas Chromatography (GC) with FID (Flame Ionization Detection)

The (free or total) sterol extract suspended in hexane is preparedaccording to procedure 1 (cf. example 5 1) a) and b)). An injectioncontrol is added to the sterol mixture, in general cholesterol at aconcentration of 10 to 50 ng/μl.

From 1 to 3 μl of samples are then injected onto a gas chromatographydevice under the following conditions. 1 to 3 μl were injected onto anAlltech SE30-type column (column reference: 30 m×0.32 mm IDX 0.25/μm).The gas vector is helium. The Split ratio is between 50 and 80. Thecolumn head pressure is 30 psi. The injector is set at 280° C. Theinitial temperature of the column is 130° C. for 0.5 of a minute. Itincreases to 230° C. at a rate of 40° C./min, and then from 230° C. to280° C. at a rate of 3° C./min. The column is then maintained at 290° C.The temperature of the detector is 310° C.

2) Gas Chromatography (GC) with FID (Flame Ionization Detection) Coupledwith Mass Spectrometry (GC/MS)

The total sterol extract suspended in hexane is prepared according toprocedure 2. The GC used is equipped with a conventional“split/splitless” injector with a conventional DB5 column that is metersin length and 0.25 mm in diameter.

The injection is carried out at 230° C. with helium as gas vector, at aflow rate of 2 ml/min. The column goes from 130 to 290° C. in 4 stages.The column is maintained at 130° C. before injection and then increasesto 230° C. with a ramp of 40° C./min, then from 230° C. to 280° C. witha ramp of 3° C./min and then from 280° C. to 290° C. with a ramp of 30°C./min. The column remains at 290° C. for 5 minutes.

At the gas chromatography column outlet, the molecules are then analyzedby means of spray mass spectrometry in an ionization chamber such asthat of a device of Turbo Mass type from Perkin Elmer. The molecules arefragmented with a high-energy electron beam. The various fragments arethen separated on a quadrupole filter and then detected on an iondetector. A mass spectrum which includes the masses of all the productsof fragmentation of the M+ ion corresponds to each mass located on theion current graph. This mass spectrum obtained at a given retention timeon the column is compared with libraries of fragmented products and alsowith those described for sterols by Quail and Kelly. (Methods inMolecular Biology Vol. 53 Yeast Protocols Edited by Evans; M. Quail andS. Kelly “The Extraction and Analysis of Sterols from Yeast” pp. 123-131(1996)).

In this way, it was possible to demonstrate the effect of the deletionof the ERG6 gene in the WGIF01 strain and in particular the absence ofergosta-8,24(28)-dienol and the presence of sterol of the type cholestahaving the double bond in the 24(25)-position.

EXAMPLE 7 Analysis of the Free and Esterified Sterols in the Yeast byHigh Performance Liquid Chromatography (HPLC) with UV Detection orDetection by Mass Spectrometry 1) Analysis by UV Detection HPLC:

Ten to 30 μl of the sterol extract (suspended in acetonitrile ormethanol and prepared according to procedure 1 or 2 (cf. example 5)) areinjected onto an X terra RP18 type 4.6×100 mm column (Waters, Milford,Mass. 01757 USA).

The separation is carried out on a gradient composed of water containing0.02% of TFA (trifluoroacetic acid) (buffer A) and of pure acetonitrile(buffer B). The column is maintained at 60° C. during the analysis.

The HPLC device used is of the “Waters 600 E System Controller” type(Waters, Milford, Mass. 01757 USA). The UV detection was carried out ona diode-array detector covering the wavelengths from 206 to 350 nm. Thecolumn was equilibrated with a buffer containing 20% (v/v) of buffer A(acetonitrile) and 80% of buffer B (water containing 0.02% of TFA(trifluoroacetic acid)). A linear gradient is formed from a solutioncontaining 50% of buffer A and 50% of buffer B. After 10 min, thecomposition of the elution buffer is 25% of buffer A for 75% of bufferB. A new linear gradient is then applied in such a way that, at 30 min,the gradient reaches the value of 100% of buffer B. This value ismaintained for 5 minutes in order to clean the column.

2) Analysis by HPLC, Detection by Mass Spectrometry (HPLC/MS):

In the case of an analysis by mass spectrometry, the sample ismaintained at 30° C. and the column is maintained at 60° C. during theanalysis. The HPLC device used is of the “Alliance HT Waters 2790” type,coupled to a “Waters MicroMass ZQ” mass detector. Unlike the precedingdetection method, the elution buffer A does not contain any TFA, but thetwo buffers A and B contain 0.01% (v/v) of formic acid.

The column was equilibrated with a buffer containing 80% of buffer A′(water containing 0.01% (v/v) of formic acid) and 20% of buffer B′(acetonitrile containing 0.01% (v/v) of formic acid).

The injection begins with a buffer containing 50% of these two buffers.A linear gradient with two ramps is formed from a solution containing50% of buffer A′ and 50% of buffer B′.

After 10 min, the composition of the elution buffer is 25% of buffer A′for 75% of buffer B′. The ramp of the gradient is then modified so as toreach 12.5% of buffer A′ and 87.5% of buffer B′ after analysis for 25minutes, and then 100% of buffer B at 30 minutes. This value ismaintained for 5 minutes in order to regenerate the column.

The “Waters MicroMass ZQ” mass detector is set for positive electrosprayionization scanning. The values of m/z are between 295 and 450. A“continuum” acquisition mode is selected for the scanning. Moreover, asignal extraction in “SIR” mode is performed in parallel at all theexpected masses by natural isotopic abundance for the sterols to beanalyzed. The detector is set so as to be able to resolve completelywithout interference from molecules differing by 1 unit of m/z. All theacquisitions are parametered in such a way that the total acquisitiontime corresponding to the scanning and to the total time for acquisitionof all the SIRs remains less than 2 seconds.

EXAMPLE 8 Culturing of the Yeast Strains for Analysis Of their SterolContent with or without ¹³C Labeling

The strains to be analyzed were cultured in a volume of 50 ml of Kappelimedium (Kappeli et al., 1985) containing 2% of normal D-glucose or ofD-glucose-U-¹³C₆ (for the labeling experiments, cf. FIG. 10).

The optical density of the starting culture is 0.1 at 600 nm. Thisculture is incubated for 72 hours at a temperature of 30° C. withshaking at 200 rpm.

The cells are then recovered by centrifugation of the medium at 600 gfor 10 minutes. The cell pellet is then analyzed directly by theanalytical techniques presented in example 5 (for the studies notrequiring induction with galactose).

However, for the studies of kinetics of induction of the expression ofdelta 7-reductase and of delta 24-reductase (strains transformed withthe plasmid pYES_Delta24 and/or pAG1), the pellet is resuspended in 50ml of fresh Kappeli medium containing 2% galactose (not labeled with ¹³Ccarbon).

This culture is incubated at a temperature of 30° C. with shaking at 200rpm.

Ten ml of culture are recovered after 0 hour, 2 hours, 4 hours, 8 hoursand 24 hours of culture.

These culture samples are centrifuged at 800 g for 10 minutes, and thecell pellet is frozen and stored at −20° C. before sterol extraction bythe methods described in example 5.

EXAMPLE 9 Identification of the Sterols Present in the Strains Analyzed

The identification of the sterols is based on the combination of thefollowing principles:

-   -   Comparison of the behavior by GC, HPLC, GC/MS and HPLC/MS with        authentic standards in the case of campesterol (ergosta-5-enol),        of ergosterol (ergosta-5,7,22-trienol), of cholesterol        (cholesta-5-enol), of desmosterol (cholesta-5,24-dienol), of        cholesta-5,22-dienol and of zymosterol (cholesta-8,24-dienol).    -   Analysis of the absorption spectrum by HPLC and diode-array UV        detection (cf. example 7-1)):

This method makes it possible to identify unambiguously, on a spectralbasis, five classes of sterols: 1) class SA1: no conjugated dienesystem, 2) class SA2: presence of a 5,7-diene system; 3) class SA3:presence of a 22,24 (25)-diene system; 4) class SA4: presence of an8,14-diene system; 5) class SA5: presence of a 22,24(28)-diene system.Classes SA3 and SA5 cannot coexist for structural reasons. Classes SA2and SA4 cannot coexist for biosynthetic reasons, class SA2 may becombined with structural units of classes SA1, SA3, SA5 so as to formadditive composite spectra.

-   -   Analysis of the retention times in GC and in HPLC on the basis        of an approximate additivity of the retention time shifts        associated with each type of unsaturation and with the presence        of a backbone of ergosta or cholesta type. Since this criterion        is not absolute, it is used as an aid to identification and in        order to lift ambiguities, but presents a risk of error if it is        used alone. It is therefore only used in combination with the        other criteria.    -   GC/MS analysis (cf. example 6-2)), which provides the molecular        mass and a fragmentation profile which can be compared to        spectral libraries.    -   HPLC/electrospray-MS analysis (cf. example 7-2)), which        provides, in the case of 3-hydroxysterols, a main signal at the        molecular mass −17 (protonation (+ 1) and loss of a water        molecule (−18)).    -   Analysis with all the above systems of the sterol composition of        various reference yeast strains disrupted at various points of        the biosynthesis.    -   Analysis of variations in the sterol composition during        complementation with various biosynthetic enzymes, and of the        kinetics of this complementation during the induction of this        complementation.    -   Analysis of the profile for labeling of the various sterols with        the carbon-13 isotope.    -   Analysis of the UV spectrum of the sterols separated by HPLC at        a given retention time. The two 5,7-conjugated double bonds        exhibit a typical spectrum with two absorption peaks between 265        and 280 nm, while the two 22,24-conjugated double bonds exhibit        an absorption peak at 235 nm. The final, 8,14-conjugated, double        bond can be identified by an absorption peak at 245 nm.

EXAMPLE 10 Identification of the Sterols Present in the BMA64 Strain

The BMA64 strain is cultured in a volume of 50 ml of Kappeli mediumcontaining 2% of D-glucose for quantitative and comparative analysis ofthe sterols.

The optical density of the starting culture is 0.1 at 600 nm. Thisculture is incubated for 72 hours at a temperature of 30° C. withshaking at 200 rpm.

The cells are then recovered by centrifugation of the medium at 600 gfor 10 minutes, and the cell pellet is analyzed by the techniquespresented in example 5. The various analyses described made it possibleto identify the sterols produced by this strain.

It was thus determined that this strain accumulates more than 80% of itsfree sterols in the form of ergosterol (ergosta-5,7,22-trienol) (cf.FIG. 8). Two other minor detectable sterols are produced by this strain;they are ergosta-5,7-dienol (substrate for the product of the ERG5 gene)(12%) and zymosterol (ergosta-8,24-dienol) (5%). No trace of cholesterolis detectable (the limit of detection of the method is approximately0.5% of observable sterols). Small amounts of lanosterol are alsodetectable (only in the analyses of total sterols).

EXAMPLE 11 Identification of the Sterols Present in the WGIF01 Strain

The WGIF01 strain (cf. example 1) was cultured in a volume of 50 ml ofKappeli medium (Kappeli et al., 1985) containing 2% of D-glucose, forthe quantitative and comparative analysis of the sterols.

The optical density of the starting culture is 0.1 at 600 nm. Thisculture is incubated for 72 hours at a temperature of 30° C. withshaking at 200 rpm.

The cells are then recovered by centrifugation of the medium at 600 gfor 10 minutes, and the cell pellet is analyzed by the techniquespresented in example 5. The various analyses described made it possibleto identify the sterols produced by this strain.

The search, in the chromatogram, for ergosta-5,7,22-trienol (ergosterol)or for ergosta-5,7-dienol is negative (less than 0.5% of the valueobtained in BMA64) in HPLC coupled to mass spectrometry. In terms of thefree sterols, the strain accumulates 50% of the total zymosterol(cholesta-8,24-dienol), substrate for the product of the ERG6 gene, and30 and 20%, respectively, of cholesta-5,7,24-trienol and ofcholesta-5,7,22,24-tetraenol probably resulting from a mechanism ofsynthesis identical to that which results in ergosta-5,7-dienol and inergosta-5,7,22-trienol in the parental strain (cf. FIGS. 3 and 8). Thisclearly shows that the biosynthetic pathway is blocked at the level oferg6 since the ERG6p enzyme (S-adenosylmethionine delta24 sterolC-methyl-transferase) converts the cholesta-8,24(25)-dienol toergosta-8,24(28)-dienol (cf. FIG. 2). This accumulation clearlyindicates that the WGIF01 strain does not possess a functional copy ofthe erg 6 gene. The results also indicate that the normal biosyntheticpathway for ergosterol in yeast, and in particular sterol 8,7-isomerase,sterol 5-desaturase and sterol delta 22-desaturase, is capable ofconverting the cholesta-type substrates with an activity which remainsconsiderable.

EXAMPLE 12 Construction of the WGIF02 Strain and Identification of theSterols Present in this Strain

The WGIF02 strain was obtained by transformation of the WGIF01 strainwith the plasmid pYES2 carrying the Δ24-reductase expression cassette(pYES_Delta24, cf. example 3). The clones were selected on a mediumlacking uracil and the presence and expression of the Δ24-reductase cDNAis verified by analyzing the sterols of these transformants by means ofprocedure 1 (cf. example 5-1)).

A clone called WGIF02 was selected since it had a sterol profile thatwas different from the WGIF01 strain; furthermore, the additional sterolhad a retention time similar to that of cholesterol (cf. FIG. 7). TheWGIF02 strain is cultured in a volume of 50 ml of Kappeli medium(Kappeli et al., 1985) containing 2% of D-glucose, for the quantitativeand comparative analysis of the sterols.

The optical density of the starting culture is 0.1 at 600 nm. Thisculture is incubated for 72 hours at a temperature of 30° C. withshaking at 200 rpm.

The cells are then recovered by centrifugation of the medium at 600 gfor 10 minutes, and the cell pellet is analyzed by the techniquespresented in example 5. The various analyses described made it possibleto identify the sterols produced by this strain.

The two sterol extract profiles for the WGIF01 strain and for the WGIF02strain are similar except for the appearance of a new peak identified,by virtue of its mass, its retention time and its conjugated doublebonds, as cholesta-5,7,22-trienol (FIGS. 2, 3 and 7). The presence ofthis compound indicates the expected presence of a 24,25-sterolreductase activity on the double bond in the 24(25)-position of thecholesta-5,7,22,24(25)-tetraenol. In addition, the amount ofcholesta-5,7,24-trienol is decreased with the appearance of thecholesta-5,7,22-trienol in the WGIF02 strain (FIGS. 7 and 8). Theactivity of the enzyme is demonstrated by the conversion ofcholesta-5,7,24-trienol representing 30% in the WGIF01 strain and only12% in WGIF02, the difference, i.e. 18%, being entirely in the form ofcholesta-5,7,22-trienol in the WGIF02 strain. This is an unexpectedresult insofar as the product of conversion of cholesta-5,7,24 by thedelta 24-reductase is cholesta-5,7, which is absent in WGIF02, andtherefore quantitatively converted to cholesta-5,7,22. This demonstratesanother unexpected result, i.e. that cholesta-5,7 is a substrate forsterol 22-desaturase, whereas cholesta-5,7,24 is, according to thesterol profile for the WGIF01 strain, a poor substrate.

EXAMPLE 13 Construction of the WGIF03 Strain and Identification of theSterols Present in this Strain

The WGIF03 strain was obtained by transformation of the WGIF01 strainwith the plasmid pAG1. This shuttle plasmid between E. coli and S.cerevisiae carries an expression cassette for Δ7-reductase, thecorresponding cDNA of which is under the control of the GAL10/CYC1promoter. The WGIF01 strain was transformed by the lithium chloridetechnique and the transformants were selected on medium containing noadenine. The expression of Delta7-reductase was verified by theappearance, in the sterol profile of the correct clones, ofcholesta-5,24(25)-dienol. A clone satisfying these criteria was moreparticularly selected and called WGIF03.

The WGIF03 strain was cultured in a volume of 50 ml of Kappeli medium(Kappeli et al., 1985) containing 2% of D-glucose, for the quantitativeand comparative analysis of the sterols.

The optical density of the starting culture is 0.1 at 600 nm. Thisculture is incubated for 72 hours at a temperature of 30° C. withshaking at 200 rpm.

The cells are then recovered by centrifugation of the medium at 600 gfor 10 minutes, and the cell pellet is analyzed by the techniquespresented in example 5. The various analyses described made it possibleto identify the sterols produced by this strain.

The expression of the delta7-sterol reductase in the WGIF01 strain (togive the WGIF03 strain), unlike the expression of the delta24-sterolreductase, results in a profound change in the sterol profile of thestrain, with a virtually complete disappearance ofcholesta-5,7,22,24-tetraenol, cholesta-7,24-dienol andcholesta-8,24-dienol.

This activity is also marked by the appearance of a major peakidentified as described previously as cholesta-5,24-dienol ordesmosterol. The amounts of cholesta-8,24-dienol change from 12 to 48%.The detected cholesta-5,7,24-trienol changes from 30 to 3% and thedetected cholesta-5,7,22,24-tetraenol changes from 23 to 4%,respectively, for the WGIF01 and WGIF03 strains. These observationsindicate, unexpectedly, that the sterol delta 7-reductase reduces thecholesta-5,7-dienol in a manner that is virtually independent of thenature of the unsaturations carried by the side chain of the sterols.This result is opposite to that observed with the sterol delta24-reductase. Unexpectedly, expression of the sterol delta 7-reductasealso results in the accumulation (12%) of a molecule that comigrateswith the cholesta-5,7-dienol. However, it appears to be relativelyunlikely, although not impossible, that this molecule ischolesta-5,7-dieneol, the theoretical level of which should decrease andnot increase under these conditions. The appearance of a small amount ofcholesta-5,22,24-trienol (8%) is also of interest. The latter sterol isthe expected product of the action of sterol 22-desaturase oncholesta-5,24-dienol, the major sterol (60%) in the WGIF03 strain(resulting from the reduction of cholesta-5,7,24-trienol by sterol delta7-reductase). The small accumulation of cholesta-5,22,24-trienolindicates, unexpectedly, that cholesta-5,24-dienol is not a goodsubstrate for sterol 22-desaturase. By virtue of the results obtained inthe WGIF02 strain (cf. example 12), it can be deduced that the presenceof an unsaturation in the 24-position (cholesta-5,24-dienol orcholesta-5,7,24-trienol) makes it difficult for the sterol 22-desaturaseto metabolize the sterols. The action of the ERG6p enzyme in convertingthe cholestas unsaturated in the 24-position to ergosta thereforeresults in the conversion of poor substrates for the ERG5 gene(22-desaturase) to good substrates.

EXAMPLE 14 Construction of the WGIF04 Strain and Identification of theSterols Present in this Strain

The WGIF04 strain was obtained by transformation of the WGIF02 strainwith the plasmid pAG1 by the lithium chloride technique, and thetransformants were selected on medium containing neither adenine noruracil. The correct transformants were then confirmed on the basis ofthe detection of cholesterol accumulation. A clone satisfying thesecriteria was more particularly selected and called WGIF04. A sample ofthe WGIF04 strain was deposited with the Collection Nationale deCultures de Microorganismes (CNCM) [National Collection of MicroorganismCultures], Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex15, France, on Apr. 22, 2004 under the registration number I-3203.

Strains that are indistinguishable from WGIF04 can also be obtained bytransforming the WGIF03 strain with pYES_Delta24 and applying the sameselection.

The WGIF04 strain was cultured in a volume of 50 ml of Kappeli mediumcontaining 2% of D-glucose, for the quantitative and comparativeanalysis of the sterols.

The optical density of the starting culture is 0.1 at 600 nm. Thisculture is incubated for 72 hours at a temperature of 30° C. withshaking at 200 rpm.

The cells are then recovered by centrifugation of the medium at 600 gfor 10 minutes, and the cell pellet is analyzed by the techniquespresented in example 5. The various analyses described made it possibleto identify the sterols produced by this strain.

Cholesterol represents 25% of the free sterols of the WGIF04 strain (cf.FIG. 9). The formation of cholesterol in this strain is independentlydemonstrated in GC and in HPLC, by comigration with an authenticstandard and by confirmation both in GC/MS and in HPLC/MS. Cholesterolis undetectable (< 0.5% of total sterols) in all the strains that do notsimultaneously express the delta 7-reductase and the delta 24-reductase.

The strains that do not have a disruption in the erg6 gene can producecholesterol; however this represents less than 5% of the total freesterols. Thus, it was possible to construct the BMA64-pYES_Delta24-pAG1strain obtained from BMA64 cotransformed with pYES_Delta24 and pAG1.This strain produces cholesterol, the latter representing a few % of thetotal sterols.

The CA10 strain was transformed with the plasmid pYES_Delta24. Thisstrain also produces cholesterol, the latter representing a few % of thetotal sterols.

It was possible to demonstrate, moreover, that the formation ofcholesterol clearly requires induction of the delta 7-reductase anddelta 24-reductase promoters (cf. FIGS. 4 and 5). The strains containingthese genes do not produce cholesterol in the absence of induction; FIG.5 indicates that the maximum level of cholesterol is reached afterapproximately 24 h of induction. In parallel with the formation ofcholesterol (cholesta-5-enol), the formation of cholesta-5,22-dienol isalso observed. Analysis of FIG. 4 and of FIG. 5 indicates that theformation of the latter compound occurs more rapidly after inductionthan the formation of cholesterol, and even begins before induction (cf.FIG. 5A: m/z=367). However, this compound is completely absent if thestrain does not carry the two plasmids pAG1 and pYES_Delta24. Theformation of 22-dehydrocholesterol is therefore a more rapid processthan that of cholesterol formation, but this process involves aprecursor which disappears rapidly after induction, leaving room for theformation of cholesterol. The cholesterol can be formed fromcholesta-5,24 via the Δ24-reductase or from cholesta-5,7 via theΔ7-reductase. Now, it has been shown that cholesta-5,7-dienol cannotaccumulate due to the fact that it is immediately converted tocholesta-5,7,22-trienol. The source of cholesterol is thereforecholesta-5,24-dienol, which is absent at the time of the induction andaccumulates at around 4 to 8 hours of induction, before decreasing ataround 24 h (FIG. 5). This explains the late appearance of cholesterolsince the prior synthesis of cholesta-5,24-dienol is required.Conversely, the two possible precursors for cholesta-5,22-dienol arecholesta-5,7,22-trienol and cholesta-5,22,24-trienol. The latter isabsent at the beginning of induction (FIG. 4), whereas the former ispresent and then rapidly decreases in parallel with the stabilization ofthe formation of cholesta-5,22-dienol. It may be concluded therefromthat the source of cholesterol is the reduction of the 5,24-dienol bythe Δ24-reductase, whereas the formation of cholesta-5,22-dienol resultsfrom the reduction of the 5,7,22-trienol by the Δ7-reductase. Theformation of cholesta-5,22-dienol by means of the action ofΔ22-desaturase on cholesterol is not completely out of the question, butappears to be a minor process on the basis of the preferentialaccumulation of cholesterol compared to cholesta-5,22-dienol at the longtime point (24 h) of the kinetics (FIGS. 4 and 5).

EXAMPLE 15 Optimization of the Biosynthetic Pathway for Cholesterol,Role of Δ22-Desaturase

For induction times of the order of 24 h for the WGIF04 strain, theaccumulation of cholesta-5,22-dienol represents approximately 50% ofthat of cholesterol in terms of free sterols (FIG. 4). Disruption of theΔ22-desaturase gene is an option for optimizing cholesterol production.The construction of a strain that is doubly disrupted in terms ofΔ22-desaturase (erg5 gene) and of the erg6 gene and that expressesΔ7-reductase and Δ24-reductase is entirely conceivable. A straincarrying the subset: disruption of Δ22-desaturase, expression ofΔ7-reductase and expression of Δ24-reductase was produced.

This strain was obtained by transformation of the CA10 strain with theplasmid pYES_Delta24 by the lithium chloride technique and by selectionfor prototrophy with respect to uracil. The strain obtained was calledCA10/Δ24.

The CA10 strain expressing Δ24 sterol reductase produces a relativelysmall amount of cholesterol (cf. FIGS. 6 and 7) and accumulates mainlyergosta-5-enol and an intermediate amount of ergosta-5,7-dienol. Theaccumulation of cholesta-5,7-dienol is very low in such a strain,indicating that disruption of the erg6 gene is essential for substantialaccumulation of derivatives of the cholesta series. The activity of theΔ24-reductase therefore, surprisingly, competes relatively little withthat of the product of the erg6 gene. It may therefore be concluded fromthese results that the simultaneous disruption of the erg5 and erg6genes is important for the purpose of optimizing the production ofcholesterol.

Disruption of the erg5 gene in the WGIF04 strain would make it possiblefor those skilled in the art to considerably increase the production ofcholesterol. The feasibility of such a strain is established by theconstruction of the WGIF04 and CA10/Δ24 strains and the fact that thisstrain is merely an obvious genetic combination to be produced from thetwo preceding strains. The data derived from WGIF04 indicate thatcholesta-5,24 disappears rapidly when Δ24-reductase is expressed(comparing the results obtained with WGIF03 and those obtained withWGIF04). This makes it possible to predict that cholesterol will besynthesized very efficiently in a strain that is simultaneouslydisrupted for the erg5 and erg6 genes and coexpresses Δ7-reductase andΔ24-reductase, the cholesterol then being the only end sterol.

In conclusion, the minimum requirement for the production of cholesterolat a threshold above or equal to 20% of the total sterols is disruptionof the erg6 gene, and expression of the Δ7-reductase and of theΔ24-reductase. Complementary disruption of Δ22-desaturase would make itpossible to improve cholesterol productivity and to eliminate theparasitic formation of cholesta-5,22-dienol as end sterol.

EXAMPLE 16 Isotope Labeling of Cholesterol and Definition of IsotopeSignatures

The principle of production of a labeled cholesterol is described inFIG. 10. This manipulation first of all consists in growing the yeast onglucose completely labeled with ¹³C, for 72 hours of culturing at 30° C.

The cells are then recovered by centrifugation of the medium at 600 gfor 10 minutes. The cell pellet is then suspended in 50 ml of freshKappeli medium containing 2% of galactose not labeled with ¹³C carbon.The cultures are stopped 2 hours, 4 hours, 8 hours or 24 hours after theswitch to galactose, and the sterols are extracted and then analyzed(cf. example 7). The switch from glucose to galactose results ininduction of the GAL10/CYC1 promoters controlling both the Δ7-reductasegene and the Δ24-reductase gene. Simultaneously, there is a change inisotope labeling of the carbon source. There ensues the de novosynthesis of metabolic intermediates and then of sterol, includingcholesterol, comprising a gradual change in labeling. This change inlabeling can be characterized by the mass profile of each intermediatesterol. In fact, the incorporation of each ¹³C atom induces a shift inmass of one atomic mass unit (AMU). Thus, for example, the cholesterolappears with a molar mass ranging from 386 to 413 daltons depending onthe degree of labeling. In analysis by HPLC-mass, using positiveelectrospray ionization, this corresponds to m/z (mass/charge) valuesranging from 369 to 396 (ion M+ H⁺—H₂O, i.e. M+1−18=385−17=369). Thesterol retention time in HPLC does not depend detectably on the degreeof labeling. The mass spectrum of an HPLC peak corresponding to a singlesterol “X” corresponds to a mass distribution which is therefore thesuperposition (the sum) of the mass distributions for the sterol “X”synthesized at various times after labeling. It is therefore a profilewhich is complex (FIG. 11), but which can be completely experimentallydetermined, and which represents a unique isotope signature whichdepends at the same time:

-   -   1) on the labeling protocol and in particular on the culture        times and conditions with ¹²C glucose and with ¹³C galactose,    -   2) on the precise genetic structure of the strain used,    -   3) on the precise time at which the cultures are stopped.

This isotope profile has several unique properties:

-   -   1) it can be modulated as desired by adjusting the culture        conditions, the strain used and the sterol chosen. A unique        label register can therefore be produced;    -   2) it is “combinable”, i.e. several isotope signatures        corresponding to several unique sterols labeled with isotope        profiles that can themselves be modulated can be combined so as        to form a “molecular alphabet”;    -   3) it is reproducible and easy to determine experimentally (cf.        FIGS. 11 and 12) the double or triple plots which indicate the        reproducibility of the profiles;    -   4) it corresponds to a molecular tracer mixture that is easy to        isolate, stable, colorless and odorless, nonvolatile and        nontoxic, and that can be incorporated into foods, a medicinal        product, additives or other products that can be assimilated by        humans;    -   5) it cannot be falsified without having the specific        recombinant strains and the very precise labeling, culturing and        extraction conditions. In addition, knowledge of the isotope        signature does not make it possible to track back to the        parameters which made it possible to produce it.

In summary, an “isotope alphabet” for general use, that cannot befalsified and that can be incorporated into products of any type,including consumables, can be readily obtained by virtue of the presentinvention. There is a virtually unlimited number of “isotope words” thatcan be constituted from such an alphabet by making use of both thelabeling profiles and the various types of sterols. The incorporation ofsuch signatures into the most varied products therefore constitutes aunique method of labeling that cannot be falsified, unlike, for example,DNA signatures, which can be reproduced once they are known. Thesignature can, moreover, be read nondestructively, for example by laserionization followed by mass spectrometry analysis (MALDI-TOF or thelike).

EXAMPLE 17 Production of Nonanimal Cholesterol Highly Labeled with ¹³C

The use of ¹³C galactose or of ¹³C ethanol and glucose instead of theunlabeled carbon sources for culturing the WGIF04 strain under theconditions described above for the sterol analyses makes it possible tosynthesize very highly labeled sterols, and in particular cholesterol(comprising at least 95% of ¹³C carbon). The preparation of¹⁴C-radioactive sterols and cholesterol is also possible by means of thesame approach. The method can also be incorporated into yeast strainsthat produce steroids, and in particular hydrocortisone (cf. patentapplication WO 02/061109), in order to produce ¹³C-labeled or¹⁴C-labeled steroids, for example for RIA assays.

EXAMPLE 18 Construction of a Strain that Produces Mainly Cholesterol

The CDR07 Mata strain is described in the patent application publishedunder the number WO 02/061109 and was deposited with the CollectionNationale de Cultures de Microorganismes (CNCM) [National Collection ofMicroorganism Cultures], rue du Docteur Roux, 75724 Paris Cedex 15,France, according to the provisions of the Treaty of Budapest at theCNCM, on Jan. 24, 2001 under the registration number I-2616.

The CDR07 MATa strain (relevant markers: ade2::GAL10/CYC1_(P)::Δ⁷;erg5::PGK1_(P)::hygro^(R); ERG6) was crossed with the WGIF01 straindescribed in example 1 (relative markers: ERG5; erg6::TRP1).

After sporulation of the diploid, the sterol composition of the sporesis determined in order to find spores that produce desmosterol, which isthe precursor for cholesterol, as described in example 6. A sporeproducing desmosterol, having a functional TRP1 gene and carrying theArabidopsis thaliana Δ7-reductase cDNA, as indicated by the PCRanalysis, was thus identified. In addition, this strain, called YIM59 issensitive to hygromycin, indicating that the ERG5 gene is functional.

A sterol preparation, prepared as described in examples 6 and 9, showedthat this YIM59 strain produces sterols that have the same retentiontime as zymosterol and desmosterol. The YIM59 strain also showedauxotrophies for adenine, leucine, uracil and histidine. The presence ofdesmosterol demonstrates that this strain expresses the sterol Δ7reductase and that it carries a nonfunctional erg6 allele.

With the aim of improving the level of expression of human DHCR24, thenucleotide sequence of the DHCR24 cDNA was modified; in order to improvethe translation at the initiating ATG, the promoter controlling theexpression of DHCR24 was also modified. The new promoter selected is theCYC1 promoter of cytochrome cl, as a replacement for the GAL1 induciblepromoter of the plasmid pYES_Delta24 (cf. example 3).

The sequence corresponding to the terminal NH2 of the DHCR24 reductaseas a fusion with the CYC1 promoter was modified as follows:

(SEQ ID No. 7) tagcgtggatggccaggcaactttagtgctgacacatacaggcatatatatatgtgtgcgacgacacatgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttatacattaggtcctttgtagcataaattactatacttctatagacacgcaaacacaaaggaattgacaagtttgtacaaaaaagcaggctaaaaaATGGAACCTGC CGTGTCGCTGGCCGTGTGCG.

The small letters represent the partial nucleotide sequence of the CYC1promoter followed by the AttB1 recombination sequences and then an AAAAsequence which precedes the initiating ATG. The sequence of the firsttwo codons was also modified (sequence GAA-CCT after the ATG initiatingcodon).

The final plasmid carrying the DHCR24 cDNA under the control of the CYC1promoter and also the S. cerevisiae 2μ origin of replication and theURA3-d selection marker was called pIM331. Its equivalent without theDHCR24 cDNA was called pIM303.

The YIM59 strain is transformed independently with the plasmids pIM303and pIM331, and two transformants carrying the plasmid pIM303(YIM59/pIM303 strain) or pIM331 (YIM59/pIM331 strain) are moreparticularly selected.

These strains are cultured in a reconstituted rich medium of the Kappelitype for 72 hours at 28° C. in order to attain an absorbance of 40 at600 nm. Total sterol extracts (esterified sterols and free sterols) ofthe YIM59/pIM303 strain (not carrying the DHCR24 cDNA) and of theYIM59/pIM331 strain (carrying the DHCR24 cDNA) are produced in thepresence of methanolic potassium hydroxide (cf. examples 5 and 6). Thesetwo strains are tested for their ability to produce cholesterol. Some ofthe results (GC) are given in FIG. 13. The retention times presented aregiven in minutes on two chromatograms.

It was thus possible to show that the strain that does not carry thevector for expression of DHCR24 (YIM59/pIM303 strain) does not producecholesterol (part A), but mainly desmosterol (part A). Conversely, thestrain that carries the DHCR24 cDNA (YIM59/pIM331 strain) produces asterol that has the retention time of cholesterol (part B). It waspossible to demonstrate, by techniques of gas chromatography coupled toelectron-impact mass spectrometry (as described in example 6), that thissterol is indeed cholesterol. Using the surface areas of each of thesterol peaks, it was possible to estimate that the amount of cholesterolproduced by the YIM59/pIM331 strain was 57% of the sterols.

Depositing of Biological Material

The following organisms were deposited, on Apr. 22, 2004, with theCollection Nationale de Cultures de Microorganismes (CNCM) [NationalCollection of Microorganism Cultures], 25 rue du Docteur Roux, 75724Paris Cedex 15, France, according to the provisions of the Treaty ofBudapest.

-   -   WGIF04 strain deposited under the registration number I-3203.

All the publications and patents mentioned are incorporated into thepresent application by way of reference.

BIBLIOGRAPHY

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TABLE 1 Evaluation of the spectral contributions of the sterolunsaturations Wavelength (nm) Unsaturation 206 235 280 5 3.9 0.0 0.0 5,7 4.0 1.7 11.5 5, 7, 22 4.8 1.7 11.5 5, 22 4.4 0.0 0.0 5, 24 6.9 0.0 0.05, 22, 24 8.7 27.0 0.0 5, 7, 24 7.5 1.7 11.5 5, 7, 22, 24 9.2 29.8 11.58, 24 6.9 0.0 0.0 (extinction coefficients mM⁻¹ cm⁻¹)

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. An organism of the kingdomFungi comprising enzyme expression of at least one of the enzymesselected from the group consisting of 7-dehydrocholesterol reductase and3β-hydroxysterol Δ24-reductase wherein said enzyme expression isobtained by transformation of the organism.
 5. (canceled)
 6. Theorganism of claim 4 comprising enzyme inactivation of at least one ofthe enzymes selected from the group consisting of C-22 sterol desaturaseand sterol 24-C-methyltransferase.
 7. (canceled)
 8. The organism ofclaim 6 wherein said enzyme inactivation is carried out by geneinactivation.
 9. (canceled)
 10. The organism of claim 4 or claim 6wherein the organism is chosen from the genus Saccharomyces orSchizosaccharomyces.
 11. A Saccharomyces cerevisiae yeast strain,characterized in that it is the WGIF04 strain deposited with theCollection Nationale de Cultures de Microorganismes (CNCM) [NationalCollection of Microorganism Cultures] on Apr. 22, 2004 under theregistration number I-3203.
 12. A method for producing cholesterol ofnonanimal origin, comprising culturing the organism of claim 4 or claim6.
 13. The method as claimed in claim 12, further comprising extractingthe cholesterol after culturing of the organism.
 14. The method asclaimed in claim 13, wherein the extraction of the cholesterol iscarried out with a non-water-miscible solvent.
 15. The method as claimedin claim 13 or 14, wherein a saponification step is carried out beforethe extraction of the cholesterol.
 16. The method as claimed in claim 13or 15, wherein a step comprising mechanical grinding of the organisms iscarried out before the saponification or the extraction of thecholesterol.
 17. (canceled)
 18. A method for producing cholesterol, orone of its metabolic intermediates, or a mixture of sterols, labeledwith ¹³C or with ¹⁴C, comprising: a) culturing an organism as claimed inclaim 4 or claim 6 on a ¹³C-labeled or ¹⁴C-labeled substrate, and b)extracting said cholesterol, or one of its metabolic intermediates, orthe mixture of sterols.
 19. A method for producing an isotopic mixturewherein the isotopic mixture is selected from the group consisting ofcholesterol, cholesterol intermediates and cholesterol metabolites,labeled at various positions using isotope labels, comprising: a)culturing an organism as claimed claim 4 or claim 6 on a labeledsubstrate, and b) removing the organism from the labeled substrate ontoan unlabeled substrate wherein the culture times on the labeledsubstrate and the unlabeled substrate produces the isotopic mixture ofcholesterol, of cholesterol intermediates or of cholesterol metabolites.20. A composition comprising an isotopic mixture wherein the isotopicmixture is selected from the group consisting of cholesterol,cholesterol intermediates and cholesterol metabolites, labeled atvarious positions using isotope labels and having a defined isotopeprofile.
 21. (canceled)
 22. A composition comprising an isotopic mixturewherein the isotopic mixture is selected from the group consisting ofcholesterol, cholesterol intermediates and cholesterol metaboliteslabeled at various positions using isotope labels, that has a definedisotope profile and that can be obtained by the method claimed in claim19.